U.S. patent number 5,296,354 [Application Number 07/726,376] was granted by the patent office on 1994-03-22 for kit for the specific assay of angiotensin ii.
This patent grant is currently assigned to Elf Sanofi. Invention is credited to Gabriel Badouaille, Jean-Alain Fehrentz, Jean Marchand, Bernard Romestand, Dominique Simon.
United States Patent |
5,296,354 |
Simon , et al. |
March 22, 1994 |
**Please see images for:
( Certificate of Correction ) ** |
Kit for the specific assay of angiotensin II
Abstract
The present invention relates to a kit for the specific assay of
Angiotension II, comprising: the anti-Ang II antibody, a solution
of labelled Ang II, solutions containing the Ang II standards at
known and increasing concentrations, the requisite washing
solution(s), a solution of anti-Ang III antibody, the said antibody
exhibiting a cross-reaction with Ang II of less than 10% and
preferably less than 5%, and, optionally, a solution of anti-Ang I
antibody and/or a solution of anti-pentapeptide antibody and/or a
solution of anti-hexapeptide antibody, the said antibodies
exhibiting a cross-reaction with Ang II of less than 10% and
preferably less than 5%.
Inventors: |
Simon; Dominique (Montpellier,
FR), Marchand; Jean (Montpellier, FR),
Badouaille; Gabriel (Pignan, FR), Romestand;
Bernard (Saint-Gely-du-Fesc, FR), Fehrentz;
Jean-Alain (Saint Nazair du Pezan, FR) |
Assignee: |
Elf Sanofi (Paris,
FR)
|
Family
ID: |
9398408 |
Appl.
No.: |
07/726,376 |
Filed: |
July 5, 1991 |
Foreign Application Priority Data
|
|
|
|
|
Jul 5, 1990 [FR] |
|
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90 08563 |
|
Current U.S.
Class: |
435/7.92;
530/387.9; 530/389.2; 436/548; 530/388.24 |
Current CPC
Class: |
C07K
16/26 (20130101); C07K 7/14 (20130101); G01N
33/74 (20130101) |
Current International
Class: |
C07K
16/26 (20060101); C07K 16/18 (20060101); C07K
7/14 (20060101); C07K 7/00 (20060101); G01N
33/74 (20060101); G01N 033/53 () |
Field of
Search: |
;435/7.9,7.91,7.92,7.93,962 ;530/387,316,387.9,388.24,389.2
;436/548,815,808 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Nussberger, J. et al. Selectivity of Angiotensin II Antisera J. of
Immunol. Methods 56(1983):85-96. .
Couraud, Pierre-Oliver Structural Analysis of the Epitopes
Recognized by Monoclonal Antibodies to Ang II, J. of Immunol.
136(9):3365-70. .
Aikawa, Tadaomi et al. Enzyme Immunoassay of Ang I Endocrinol.
105(1):1-6 (1979). .
Takai, E. et al. a solid-phase enzyme immunoassay for determination
of IgM and IgG Ab against translation products of pre-S1 and pre-S2
regions of hepatitis B virus-J. Immunol. Meth. 23:23-30 (1986).
.
Nussberger, J. et al., "A Simplified Radioimmunoassay for
Physiologically Active Agniotensin Peptides [(1-8) Octa-and (2-8)
Heptapeptides]", Horm. metabol. Res. 16 (1984):606-610..
|
Primary Examiner: Kepplinger; Esther L.
Assistant Examiner: Wortman; Donna C.
Attorney, Agent or Firm: Foley & Lardner
Claims
We claim
1. A kit for the specific assay of Angiotensin II, comprising:
(a) an anti-Angiotensin II antibody,
(b) a solution of labelled Angiotensin II, and
(c) a solution of anti-Angiotensin III antibody, said
anti-Angiotensin III antibody exhibiting a crossreactivity with
Angiotensin II of less than 10%.
2. A kit according to claim 1, wherein said Angiotensin II is
labelled with an enzyme.
3. A kit according to claim 2, which further comprises a solution
containing a substrate for said enzyme labelling said Angiotensin
II.
4. A kit according to claim 3, further comprising a reagent for
visualizing the activity of said enzyme and a solution capable of
stopping the reaction of said enzyme and said substrate.
5. A kit according to claim 1, wherein said Angiotensin II is
labelled with a label selected from the group consisting of a
fluorescent compound, a luminescent compound and a
radioisotope.
6. A kit according to claim 1, wherein said Angiotensin II antibody
is monoclonal.
7. A kit according to claim 1, wherein said Angiotensin II antibody
is in solution.
8. A kit according to claim 1, wherein said Angiotensin II antibody
is bound to a solid support.
9. A kit according to claim 8, wherein said solid support is a
tube, a microtitration plate or particles.
10. A kit according to claim 9, wherein said particles are
magnetic.
11. A kit according to claim 1, wherein said anti-Angiotensin III
antibody is a monoclonal antibody.
12. A kit according to claim 1, wherein said anti-Angiotensin II
antibody is a polyclonal rabbit antiserum.
13. A kit according to claim 1, wherein said anti-Angiotensin III
antibody exhibits a crossreactivity with Angiotensin II of less
than 5%.
14. A kit according to claim 1, further comprising at least one
additional antibody selected from the group consisting of an
anti-Angiotensin I antibody, an antibody that binds to hexapeptide
IV and an antibody that binds to pentapeptide V, wherein each said
additional antibody exhibits a cross-reactivity with Angiotensin II
of less than 10%.
15. A kit according to claim 14, wherein each said additional
antibody exhibits a crossreactivity with Angiotensin II of less
than 5%.
16. A kit according to claim 14, wherein said additional antibody
is a monoclonal antibody.
17. A kit according to claim 14, wherein said additional antibody
is a polyclonal rabbit antiserum.
18. A kit according to claim 1, further comprising a plurality of
standard solutions each including a different predetermined amount
of Angiotensin II, said amount being greater than or equal to
zero.
19. A kit according to claim 1, further comprising a washing
solution.
20. A kit according to claim 1, wherein said anti-Angiotensin II
antibody is polyclonal.
21. A kit according to claim 1, wherein said anti-Angiotensin III
antibody is obtained by immunization of an animal with a peptide
derivative of formula (amino acids 1-7 or 2-7 or 3-7 of SEQUENCE ID
NO. 2)
in which
R.sub.1 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n
--CO--,
n denotes an integer from 1 to 9, and
X.sub.1 denotes a dipeptide Arg-Val residue.
22. A kit according to claim 21, further comprising
(d) at least one additional antibody selected from the group
consisting of
(i) an anti-Angiotensin I antibody obtained by immunization of an
animal with a peptide derivative of formula (SEQUENCE ID NO. 3)
H-Cys-R.sub.2 -R.sub.3 -Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH
in which
R.sub.3 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n --CO--,
and
n denotes an integer from 1 to 9 inclusive,
(ii) an anti-pentapeptide antibody obtained by immunization of an
animal with a peptide derivative of formula (amino acids 1-7 or 2-7
or 3-7 of SEQUENCE ID NO. 2) H-X.sub.1 -Tyr-Ile-His-R.sub.1
-R.sub.2 -Cys-NH.sub.2
in which
R.sub.1 denotes a direct bond or a chain-link comprising 1 to 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n
--CO--,
n denotes an integer from 1 to 9 inclusive, and
X.sub.1 denotes a direct bond,
and
(iii) an anti-hexapeptide antibody obtained by immunization of an
animal with a peptide derivative of formula (amino acids 1-7 or 2-7
or 3-7 of SEQUENCE ID NO. 2) H-X.sub.1 -Tyr-Ile-His-R.sub.1
-R.sub.2 -Cys-NH.sub.2
in which
R.sub.1 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n
--CO--,
n denotes an integer from 1 to 9 inclusive, and
X.sub.1 denotes a valine residue,
said additional antibody exhibiting a crossreactivity with
Angiotensin II of less than 10%.
23. A kit according to claim 22, wherein said anti-Angiotensin III
exhibits a crossreactivity with Angiotensin II of less than 5%.
24. A kit according to claim 22, wherein said additional antibody
exhibits a crossreactivity with Angiotensin II of less than 5%.
25. A kit according to claim 21, wherein said anti-Angiotensin II
antibody exhibits a crossreactivity with Angiotensin II of less
than 5%.
26. An anti-Angiotensin III antibody obtained by immunization of an
animal with a peptide derivative of formula (amino acids 1-8 of
SEQUENCE ID NO. 2)
in which
R.sub.1 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n --CO--,
and
n denotes an integer from 1 to 9 inclusive,
wherein said peptide derivative is bound to a carrier protein, and
wherein said anti-Angiotensin III antibody is specific for
Angiotensin III and exhibits a cross-reactivity with Angiotensin II
of less than 10%.
27. An anti-Angiotensin III antibody according to claim 26, wherein
in said peptide derivative
R.sub.1 denotes Pro or Pro bound to another natural amino acid, and
n=5.
28. An anti-angiotensin III antibody according to claim 27, which
is a polyclonal rabbit antiserum and which exhibits a
crossreactivity with Angiotensin II of less than 1%.
29. An anti-Angiotensin III antibody according to claim 13, wherein
said anti-Angiotension III antibody exhibits a crossreactivity with
Angiotensin II of less than 5%.
30. An anti-pentapeptide antibody obtained by immunization of an
animal with a peptide derivative of formula (amino acids 3-8 of
SEQUENCE ID NO. 2)
in which
R.sub.1 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n --CO--,
and
n denotes an integer from 1 to 9 inclusive,
wherein said peptide derivative is bound to a carrier protein, and
wherein said anti-pentapeptide antibody is specific for a
degradation pentapeptide having all but the two N-terminal amino
acid residues of Angiotensin III, and exhibits a crossreactivity
with Angiotensin II of less than 10%.
31. An anti-pentapeptide antibody according to claim 30, wherein in
said peptide derivative
R.sub.1 denotes Pro or Pro bound to another natural amino acid, and
n=5.
32. An anti-pentapeptide antibody according to claim 31, wherein
said anti-pentapeptide antibody exhibits a crossreactivity with
Angiotensin II of less than 1%.
33. An anti-pentapeptide antibody according to claim 30, wherein
said anti-pentapeptide antibody exhibits a crossreactivity with
Angiotensin II of less than 5%.
34. An anti-hexapeptide antibody obtained by immunization of an
animal with a peptide derivative of formula (amino acids 2-8 of
SEQUENCE ID NO. 2)
in which
R.sub.1 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n --CO--,
and
n denotes an integer from 1 to 9 inclusive,
wherein said peptide derivative is bound to a carrier protein, and
wherein said anti-hexapeptide antibody is specific for a
degradation hexapeptide having all but the N-terminal amino acid
residue of Angiotensin III, and exhibits a cross-reactivity with
Angiotensin II of less than 10%.
35. An anti-hexapeptide antibody according to claim 34, wherein in
said peptide derivative
R.sub.1 denotes Pro or Pro bound to another natural amino acid, and
n=5.
36. An anti-hexapeptide antibody according to claim 34, wherein
said anti-hexapeptide antibody exhibits a crossreactivity with
Angiotensin II of less than 5%.
37. An anti-Angiotensin I antibody obtained by immunization of an
animal with a peptide derivative of formula (SEQUENCE ID NO.
3):
in which
R.sub.3 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n --CO--,
and
n denotes an integer from 1 to 9 inclusive,
wherein said peptide derivative is bound to a carrier protein, and
wherein said anti-Angiotensin I antibody is specific for
Angiotensin I and exhibits a cross-reactivity with Angiotensin II
of less than 10%.
38. An anti-Angiotensin I antibody according to claim 37, wherein
in said peptide derivative
R.sub.3 denotes Asp or Asp bound to another natural amino acid, and
n=5.
39. An anti-Angiotensin I antibody according to claim 37, wherein
said anti-Angiotensin I antibody exhibits a cross-reactivity with
Angiotensin II of less than 5%.
40. A method for the assay of Angiotensin II which comprises the
steps of:
a) incubating
i) an anti-Angiotensin II antibody with a reaction medium
comprising
ii) an extract of a biological fluid,
iii) labeled Angiotensin II, and
iv) an anti-Angiotensin III antibody which is specific for
Angiotensin III and exhibits a crossreactivity with Angiotensin II
of less than 10%, at a temperature between 4.degree. C. and room
temperature for 12 to 72 hours;
b) separating labeled and unlabeled Angiotensin II bound to said
anti-Angiotensin II antibody from said reaction medium; and
c) measuring the amount of labeled Angiotensin II bound to said
anti-Angiotensin II antibody to determine the amount of unlabeled
Angiotensin II present in said extract.
41. A method according to claim 40, wherein said anti-Angiotensin
II antibody is bound to particles.
42. A method according to claim 41, wherein said particles are
magnetic.
43. A method according to claim 40, wherein said extract is a
plasma extract.
44. A method according to claim 40, wherein said anti-Angiotensin
III antibody is obtained by immunization of an animal with a
peptide derivative of formula (amino acids 1-8 of SEQUENCE ID NO.
2)
in which
R.sub.1 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids,
R.sub.2 denotes a group of formula --NH--(CH.sub.2).sub.n --CO--,
and
n denotes an integer from 1 to 9 inclusive,
wherein said peptide derivative is bound to a carrier protein.
45. A method according to claim 40, wherein said reaction medium
further comprises: (v) one or more antibodies selected from this
group consisting of an antibody that binds to pentapeptide V, an
antibody that binds to hexapeptide IV and an anti-Angiotensin I
antibody, wherein each of said one or more antibodies exhibit a
cross-reactivity with Angiotensin II of less than 10%.
46. A method according to claim 40, wherein said determination in
step c) is effected by reference to a calibration curve.
47. A method according to claim 46, wherein said calibration curve
is obtained by:
(A) incubating
i) an anti-Angiotensin II antibody with a plurality of reaction
media each comprising
ii) an solution including a different predetermined amount of
Angiotensin II, said amount being greater than or equal to
zero,
iii) said labeled anti-Angiotensin II, and
iv) said anti-Angiotensin III antibody, at a temperature between
4.degree. C. and room temperature for 12 to 72 hours;
(B) separating said Angiotensin II bound to said anti-Angiotensin
II antibody from said Angiotensin II remaining unbound; and
(C) measuring the amount of labeled Angiotensin II bound to said
anti-Angiotensin II antibody in each of said plurality of reaction
media and relating each said amount to the amount of unlabeled
Angiotensin II employed in the respective reaction medium to yield
said calibration curve.
48. An antibody that binds to hexapeptide IV, wherein said antibody
exhibits a cross-reactivity with Angiotensin II of less than
10%.
49. An antibody that binds to pentapeptide V, wherein said antibody
exhibits a cross-reactivity with Angiotensin II of less than 10%.
Description
The present invention relates to a kit for the specific immunoassay
of angiotension II and to the method for the immunoassay of
angiotension II using the said kit.
Angiotension II is a potent vasopressor agent which is the
biologically active product of the renin-angiotension system: renin
acts on the angiotensinogen of the plasma to produce a decapeptide
(1-10), angiotensin I, the latter is converted to an octapeptide
(1-8), angiotensin II, by the action of the converting enzyme and
an aminopeptidase then produces a heptapeptide (2-8): angiotensin
III.
The formulae of these different peptides in mammals are given
below:
Angiotensin I, hereinafter designated Ang I (SEQUENCE ID NO. 1):
##STR1##
Angiotensin II, hereinafter designated Ang II (amino acids 1-8 of
SEQUENCE ID NO. 1): ##STR2##
Angiotensin III,hereinafter designated Ang III (amino acids 2-8) of
SEQUENCE ID NO. 1): ##STR3##
A recent publication has shown that Ang III is itself degraded to
two peptides (D. J. CAMBELL et al., J. Hypertens., 1990, 8,
165-172). Among these degradation peptides, special mention may be
made of a hexapeptide of formula (amino acids 4-8 of SEQUENCE ID
NO. 1): ##STR4## hereinafter designated hexapeptide IV, and a
pentapeptide of formula (amino acids 4-8 of SEQUENCE ID NO. 1):
##STR5## hereinafter designated pentapeptide (V).
These degradation peptides are present in human plasma. It is
probable that they are also to be found in the plasma of other
mammals.
Exploration of the hormonal aspects of the renin-angiotensin system
is very important for the diagnosis and treatment of arterial
hypertension. At the present time, this exploration performed in
the clinical situation is limited to measurement of the enzymatic
activity of plasma renin and of the activity of the converting
enzyme and to measurement of the plasma concentration of active
renin.
The direct assay of Ang II, which is the active peptide of the
system, is difficult to carry out for several reasons:
very low plasma concentration of Ang II (3 to 20 pg/ml in normal
subjects, equivalent to 3.times.10.sup.-12 to 2.times.10.sup.-11
M), which necessitates the use of a very high-affinity anti-Ang II
antibody
structural similarity of Ang II to various peptides derived from
the cleavage of angiotensinogen and present in plasma, especially
Ang I and above all Ang III or other degradation peptides which can
interfere in the assay.
Thus, J. NUSSBERGER et al. describe a radioimmunoassay which does
not permit a distinction to be made between Ang II and III in human
plasma (Hormon. Metab. Res., 1984, 16 (II), 606-610 and J.
Hypertens., suppl., 1988, 6 (4), S424-S425). Moreover, European
Patent Application 273,453 describes several anti-Ang II monoclonal
antibodies, all of which exhibit a cross-reactivity with Ang III;
the lowest cross-reactivity, observed with one of them, is 32%.
Thus, the reaction of Ang II with this antibody is not
specific.
Furthermore, this monoclonal antibody has a low affinity, of the
order of 10.sup.9 M.sup.-1 which is insufficient to permit an assay
in plasma (Biochem. Biophys. Res. Commun. 1987, 143, p.
133-139).
The anti-Ang II antibody referred to as 4D8 and described in the
proceedings of the Congress "The Renin-Angiotensin System as a
Therapeutic Target" Basle, 29-31 Oct. 1989, a SANOFI communication,
possesses a strong affinity of the order of 10.sup.-11 M.sup.-1 ;
but it exhibits a cross-reactivity of the order of 100% with Ang
III and with the hexapeptide (IV); it exhibits an approximately 50%
cross-reactivity with the pentapeptide (V). In fact, it is very
difficult, and unknown at the present time, to have an antibody
both with a very high affinity for Ang II and not exhibiting a
cross-reaction with Ang III and the degradation peptides.
Moreover, the specific assay of Ang II presents, at the present
time, a methodological problem which necessitates performing
several steps in succession on the blood sample (J. NUSSBERGER et
al., Hypertension, 1986, 8, 476-482):
1. an extraction of the different angiotensins and of the
degradation peptides to prepare a plasma extract containing
them,
2. a separation by high performance liquid chromatography (HPLC) of
the different angiotensins derived from the metabolism of
angiotensinogen and present in plasma,
3. a radioimmunoassay of the fractions collected after elution.
Extraction of the angiotensins from the plasma is an essential step
common to all the techniques of assay of Ang II; it is performed by
chromatography on a silica column according to the method described
by J. NUSSBERGER et al. in Hypertension, 1985 (7), suppl. 1,
I-1-I-7.
As a result of the step of separation by HPLC, this technique is
cumbersome and tedious to carry out; for this reason, it cannot be
used to assay a large number of plasma samples under routine
conditions.
An attempt has been made to develop a specific assay for Ang II,
enabling the level of Ang II to be measured accurately irrespective
of the concentrations of Ang I, Ang III and the degradation
peptides present in the medium, and which can be carried out in a
time compatible with routine clinical use. Such a specific assay is
of great interest:
either for studying the physiology of the renin-angiotensin system
in different animal species in experimental situations,
or for diagnosing pathological situations in man,
or for the diagnosis and monitoring of treatments for arterial
hypertension.
Generally, Ang II is assayed from plasma. It is possible, according
to the present invention, to carry out the assay of Ang II either
in plasma or in any other biological fluid in which it is
present.
The assay according to the invention may be performed with any
animal species in which it is desired to find out the specific
measurement of Ang II, and more especially in man.
According to the present invention, it has been found that a
specific immunoassay of Ang II may be performed by using
simultaneously an antibody directed towards Ang II, intended for
the actual assay, and an antibody specific for Ang III as well as,
optionally , one or more antibodies each specific for Ang I or for
a degradation peptide. Antibody specific for Ang III and antibody
specific for Ang I or for a degradation peptide are understood to
mean antibodies each exhibiting a cross-reaction with respect to
Ang II of less than 10%. Thus, the Ang III and, optionally, the Ang
I and the degradation peptides, each captured by a specific
antibody, are in practice no longer available to give
cross-reactions with the anti-Ang II antibody. The Ang II assay
thus becomes completely specific, that is to say completely free
from all interference due to the presence of related peptides in
the sample.
According to the present invention, the measurement of Ang II
entails only two steps:
1. extraction of the different angiotensins and of the degradation
peptides to prepare the extract of biological fluid containing
them,
2. the actual immunoassay of the Ang II contained in the said
extract.
Thus, the separation by HPLC of the different angiotensins in the
extract is avoided, this step being the slowest and most laborious
of the protocol for specific assay of Ang II.
The immunoassay used is a competitive assay: labelled Ang II bound
to the anti-Ang II antibody is displaced by the unlabelled Ang II
contained in the sample to be assayed. Reference to a calibration
curve, established with standards containing known quantities of
Ang II, enables the Ang II concentration present in the sample to
be determined.
The Ang II is labelled either with a radioelement such as tritium
(.sup.3 H) or iodine-125 ( .sup.125 I), or with an enzyme such as,
for example, acetylcholinesterase, peroxidase, alkaline phosphatase
or .beta.-galactosidase, or with a fluorescent label, or with a
luminescent label.
According to the present invention, an antibody specific for Ang
III and, optionally, one or more antibodies each specific for Ang I
or for a degradation peptide, which mask the Ang III and,
optionally, the Ang I and the degradation peptides present in the
sample by formation of the corresponding antigen-antibody
complex(es), are added to the reaction medium. The appearance of
cross-reactions between the Ang III and, optionally, the Ang I and
the degradation products, present in the sample, with the anti-Ang
II antibody is thereby avoided.
The subject of the present invention is also a kit for the specific
assay of Ang II, comprising:
the anti-Ang II antibody,
a solution of labelled Ang II,
solutions containing the Ang II standards at known and increasing
concentrations,
the requisite washing solution(s),
a solution of anti-Ang III antibody, the said antibody exhibiting a
cross-reaction with Ang II of less than 10% and preferably less
than 5%,
and, optionally, a solution of anti-Ang I antibody and/or a
solution of anti-hexapeptide (IV) antibody and/or a solution of
anti-pentapeptide (V) antibody, the said antibodies exhibiting a
cross-reaction with Ang II of less than 10% and preferably less
than 5%.
When the Ang II is labelled with an enzyme, the kit comprises, in
addition:
a solution containing the substrate for the said enzyme and,
optionally one or more reagents necessary for visualising the
activity of the enzyme,
a solution intended for stopping the enzymatic reaction.
The anti-Ang II antibody used in the assay kit can be polyclonal or
monoclonal; it may be used in solution or bound to a solid support.
As solid supports, tubes, microtitration plates and particles,
magnetic or otherwise, may be mentioned.
According to the present invention, the preferred anti-Ang II
antibody is monoclonal; advantageously, it is bound to a solid
support, according to methods well known to those skilled in the
art.
The anti-Ang III antibody can be a polyclonal antibody or a
monoclonal antibody. The same applies to the anti-Ang I,
anti-hexapeptide (IV) and anti-pentapeptide (V) antibodies.
When the antibodies used are in solution, the appropriate dilution
for each of the peptides, present in the sample to be assayed, to
be captured by the corresponding anti-peptide antibody is
determined prior to the assay.
The subject of the present invention is also the method in which
the assay kit according to the invention is used. The said method
is characterised in that it comprises the following steps:
a) the reaction medium comprising the following is prepared:
the extract of biological fluid to be assayed,
the anti-Ang II antibody,
the labelled Ang II,
the anti-Ang III antibody,
and, optionally, the anti-Ang I antibody and/or the
anti-hexapeptide (IV) antibody and/or the anti-pentapeptide (V)
antibody;
b) the reaction media useful for the calibration, each comprising
the following, are prepared simultaneously:
an Ang II standard,
the anti-Ang II antibody,
the labelled Ang II,
the anti-Ang III antibody,
and, optionally, anti-Ang I antibody and/or the anti-hexapeptide
(IV) antibody and/or the anti-pentapeptide (V) antibody;
c) the media are left to incubate at a temperature between
4.degree. C. and room temperature for 12 to 72 hours;
d) the Ang II bound to the anti-Ang II antibody is separated from
the free Ang II;
e) lastly, the reading of the results is performed according to a
suitable method.
Preferably, the method according to the invention is used for the
specific assay of Ang II in a plasma extract.
When the anti-Ang II antibody is used in solution, steps a) and b)
are carried out by mixing:
25 to 500 .mu.l of extract of biological fluid to be assayed, or of
Ang II standard, with a medium containing:
25 to 500 .mu.l of solution of anti-Ang II antibody,
25 to 500 .mu.l of solution of anti-Ang III antibody,
optionally, 25 to 500 .mu.l of solution of anti-Ang I antibody
and/or 25 to 500 .mu.l of anti-hexapeptide (IV) antibody and/or 25
to 500 .mu.l of anti-pentapeptide (V) antibody, and
25 to 500 .mu.l of solution of labelled Ang II.
It is possible, where appropriate, to perform a 3- to 24-hour
preincubation before adding the solution of labelled Ang II.
Step d) is carried out by a conventionally used separating
method:
either by immunological binding using antibodies directed towards
the immunoglobulins of the animal species to which the anti-Ang II
antibody used belongs, these antibodies themselves being
insolubilised on a solid phase (insoluble support such as, for
example, tubes, microtitration plates or particles, magnetic or
otherwise),
or by physicochemical precipitation of the antigen-antibody complex
using a precipitating agent such as polyethylene glycol,
or by separation of the free antigen with the charcoal-dextran
complex (J. NUSSBERGER et al., J. Lab. Clin. Med., 1984, 103 (2),
304-312).
When, according to another embodiment of the method according to
the invention, the anti-Ang II antibody is bound to a solid
support, the preparation of the reaction medium (steps a and b) is
performed in the presence of the said support. It is possible, for
example, to use tubes coated with anti-Ang II antibodies in which
the following are mixed:
25 to 500 .mu.l of extract of biological fluid to be assayed or of
Ang II standard,
25 to 500 .mu.l of solution of anti-Ang III antibody,
optionally, 25 to 500 .mu.l of solution of anti-Ang I antibody
and/or 25 to 500 .mu.l of anti-hexapeptide (IV) antibody and/or 25
to 500 .mu.l of anti-pentapeptide (V) antibody, and
25 to 500 .mu.l of solution of labelled Ang II.
According to this embodiment of the method according to the
invention, the separation (step d) is carried out by aspiration of
the incubation solution from the reaction medium, followed by
washing and further aspiration in order to remove Ang II which is
not complexed by the antibody bound to the solid support. It is
also possible to prepare the mixture described above in a normal
tube or vessel and to add the anti-Ang II antibody in the form of
particles, magnetic or otherwise, to which the anti-Ang II antibody
is bound. In this case, the separation (step d) is carried out
according to the same principle as above, but by application of a
method enabling the particles to be retained (centrifugation,
filtration, use of a magnet where magnetic particles are
involved).
In all cases, the reading step e), which is applied to the solid
phase in which the anti-Ang II antibody complexing the Ang II to be
assayed is included, or to which this antibody is bound, is then
performed directly.
Generally speaking, the reading step depends on the method of
labelling used for the Ang II. Thus:
either a measurement of radioactivity, when radiolabelling has been
used,
or a measurement of absorbance or of light emission, when
fluorescent or luminescent labelling or enzyme labelling, depending
on the nature of the enzyme and substrate employed, has been used,
is performed.
In the description which follows and in the claims, the amino acids
are named using the abbreviations recommended by the IUPAC; in
addition, the following abbreviations are used:
BOP benzotriazolyloxytris(dimethylamino)phosphonium
hexafluorophosphate
Boc: tert-butoxycarbonyl
Mob: para-methoxybenzyl
DCB: 2,6-dichlorobenzyl
Tos: tosyl
MAb: monoclonal antibody
BSA: bovine serum albumin
BGG: bovine gamma-globulin
cpm: counts per minute
PEG: polyethylene glycol
LPH: Limulus polyphemus haemocyanin
Sulpho-MBS: N-(3-maleimidobenzoyloxy)sulphosuccinimide sodium
salt
Bo: concentration of the (labelled Ang II)-anti-Ang II antibody
complex in the absence of unlabelled Ang II or unlabelled Ang
III,
B: concentration of the (labelled Ang II)-anti-Ang II antibody
complex for each concentration of unlabelled Ang II or unlabelled
Ang III.
Lastly, another subject of the present invention is the preparation
of anti-Ang III antibodies.
The anti-Ang III antibody is prepared in a conventional manner by
immunisation of an animal with an immunogen. Said immunogen
consists of a peptide derivative bound to a modified protein or
carrier protein (see Am. J. Obstet. Gyneco. 1972, 113, p. 751-757).
As examples of animals capable of being used in this method,
rabbits, goats, sheep, rats and mice may be mentioned.
The monoclonal antibodies are obtained by the method of Kohler and
Milstein which is well known to those skilled in the art.
In the same manner, anti-Ang I, anti-hexapeptide (IV) and
anti-pentapeptide (V) antibodies may be prepared.
The use of a carrier protein in the immunisation in relation to
molecules of low molecular weight (hapten) enables the formation of
specific anti-hapten antibodies to be induced in the animal.
The most widely used carrier proteins are, at the present time,
albumins of animal origin (commercially available, stable, highly
immunogenic). The synthesis of the protein-peptide conjugate is
carried out by the formation of a thioether type covalent bond
between the thio group of the peptide and the maleimide group of
the protein, introduced beforehand by reaction with sulpho-MBS.
According to the present invention, the peptide derivative chosen
as an immunogen for preparing the anti-Ang III antibody corresponds
to the formula (SEQUENCE ID NO. 2):
in which
R.sub.1 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids. Preferably, R.sub.1 represents -Pro- or --Pro-
bound to another natural amino acid;
R.sub.2 represents a group of formula: --NH--(CH.sub.2).sub.n
--CO-- in which n varies between 1 and 9; preferably n is 5.
Thus, according to the present invention, the preferred anti-Ang
III antibody is the antiserum obtained by immunisation of an animal
with the immunogen consisting of the peptide (VI) bound to a
carrier protein.
More especially preferred is the antiserum obtained with the
immunogen consisting of the peptide of formula (6-aminohexanoic
acid species of SEQUENCE ID NO. 2):
in which R'.sub.1 represents -Pro- or Pro- bound to another amino
acid and Ahx denotes a 6-aminohexanoic acid residue, the said
peptide being carried by bovine serum albumin modified with
sulpho-MBS. When the animal is a rabbit, the antiserum thereby
obtained exhibits a cross-reaction with Ang II of less than 1%.
Likewise, according to the present invention, the peptide
derivative chosen as an immunogen for preparing the anti-Ang I
antibody corresponds to the formula (SEQUENCE ID NO. 3):
in which:
R.sub.3 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids. Preferably, R.sub.3 represents Asp or Asp
bound to another natural amino acid;
R.sub.2 has the meaning stated above for (VI).
According to the present invention, the peptide derivative chosen
as an immunogen for preparing the anti-hexapeptide antibody
corresponds to the formula (amino acids 2-8 of SEQUENCE ID NO.
2):
in which R.sub.1 and R.sub.2 have the meanings stated above for
(VI).
Lastly, according to the present invention, the peptide derivative
chosen as an immunogen for preparing the anti-pentapeptide antibody
corresponds to the formula (amino acids 3-8) of SEQUENCE ID NO.
2):
in which R.sub.1 and R.sub.2 have the meanings stated above for
(VI).
In the definitions of R.sub.1, R'.sub.1 and R.sub.3, the other
natural amino acid may be chosen from the following amino acids:
Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Cys, Tyr.
Thus, according to the present invention, the antibodies are
obtained by immunisation of an animal with a peptide derivative of
formula (VII) or a peptide derivative of formula (amino acids 1-7
or 2-7 or 3-7 of SEQUENCE ID NO. 2):
in which:
R.sub.1 denotes a direct bond or a chain-link comprising 1 or 2
natural amino acids. Preferably, R.sub.1 represents -Pro- or --Pro-
bound to another natural amino acid;
R.sub.2 represents a group of formula --NH--(CH.sub.2).sub.n --CO--
in which n varies between 1 and 9; preferably n is 5, and
characterised in that:
when X.sub.1 represents a direct bond, the antibody is specific for
the pentapeptide,
when X.sub.1 represents a valine residue, the antibody is specific
for the hexapeptide,
when X.sub.1 represents a residue of the dipeptide Arg-Val, the
antibody is specific for angiotensin III.
The antibodies according to the invention exhibit a cross-reaction
with Ang II of less than 10% and preferably less than 5%.
The above peptide derivatives employed for the production of the
antibodies according to the invention constitute another subject of
the invention.
Said peptides are prepared by conventional peptide synthesis
methods, notably by the so-called step-wise process, preferably in
solid phase.
The invention will now be described in greater detail by means of
the illustrative examples below.
EXAMPLE 1
Preparation of anti-Ang III.
A) Preparation of the peptide (VI) in which R.sub.1 =Pro and
R.sub.2 =Ahx.
The compound VI was synthesised in the solid phase using methods
known in peptide chemistry.
The solid phase used is 4-methylbenzhydrylamine resin
functionalised to a level of 0.4 mmol per gram. The successive
condensation of amino acids is performed with the BOP reagent; the
alpha-amino functions are protected temporarily with a Boc group
and deprotected with a trifluoroacetic
acid/dichloroethane/ethanedithiol (35:70:5 v/v/v) solution. The
side chains of the different amino acids are protected with
suitable groups: Mob for Cys, Boc for His, DCB for Tyr, Tos for
Arg.
Once synthesised, the peptide is deprotected and separated from the
resin by the action of hydrofluoric acid containing 10% of anisole
for 1 hour at 0.degree. C. The peptide is then precipitated with
ether, taken up with an acetonitrile/water/trifluoroacetic acid
(60:40:0.1 v/v/v) solution and lyophilised. Purification is
performed by liquid chromatography under pressure on a
reversed-phase column.
The various analytical results are in agreement with the expected
structure for the peptide (VI).
B) Preparation of the modified protein (LERNER, R. A. et al. (1981)
Proc. Nat. Acad. Sci. USA, 78, 3403-3407).
The protein chosen for binding the peptide (VI) is bovine serum
albumin (BSA) modified by reaction with sulpho-MBS.
22.4 mg of solid sulpho-MBS (Pierce) are added to a solution of 4
ml of BSA (containing 10 mg/ml) in 0.05M potassium phosphate buffer
pH 8.0, equivalent to a stoichiometry of 80MBS/BSA equivalents. The
reaction is allowed to proceed for 1 hour at room temperature with
. gentle stirring and the mixture is then dialysed for 18 hours at
4.degree. C. against 2 liters of 0.1M phosphate buffer pH 7.0.
Determination of the degree of substitution is carried out by
measuring the number of maleimide groups introduced by means of
reaction with cysteine radiolabelled with .sup.14 C. 0.6 ml of 0.1M
phosphate buffer pH 7.0 and 0.05 ml of 20 mM cysteine to which
.sup.14 C has been added (final concentration 3 kBq/.mu.mol of
cysteine) are added to 0.4 ml of reaction medium. After incubation
for 1 hour 30 minutes at 30.degree. C. followed by filtration of
the reaction medium through a chromatography column under suitable
conditions, measurement of the radioactivity enables the observed
degree of modification to be calculated, which is 11 maleimide
groups per mol of BSA.
C) Coupling of the peptide (VI).
5 mg of peptide (VI) prepared above are dissolved in 0.25 ml of
distilled water. This solution is added to 1 ml of modified BSA
solution (10 mg/ml). The mixture is incubated for 3 hours at room
temperature and 18 hours at 4.degree. C., and several extensive
dialyses are then performed against 0.1M phosphate buffer pH
7.0.
The test with radiolabelled cysteine is carried out again on the
protein after coupling of the peptide. Determination of the number
of unmodified maleimide groups indicates by difference a degree of
substitution of 7 mol of peptide (VI) per mole of BSA.
Two other immunogenic conjugates using Limulus polyphemus
haemocyanin (LPH) and human transferrin (SIGMA) as carrier proteins
were prepared under the same conditions.
D) Preparation of anti-Ang III antibody (or antiserum).
The animal used is the New Zealand breed rabbit. The first
immunisation consists in injecting 1 mg of immunogen intradermally
in the presence of Freund's complete adjuvant. Boosters are
performed subcutaneously at 1-month intervals with the same
quantity of immunogen in the presence of Freund's complete
adjuvant.
E) Determination of the titre of the anti-Ang III sera.
100 .mu.l of a dilution of rabbit serum in 0.1M imidazole-HCl
buffer pH 7.4+0.2% BSA are incubated in the presence of 100 .mu.l
of iodine-125-labelled Ang III having a specific activity of 74
TBq/mmol (Amersham), providing 5000 cpm.
Separation of the Ang III bound to the antibody from free Ang III
is carried out by adding 1 ml of 20% polyethylene glycol 6000 (PEG)
and 100 .mu.l of solution of bovine globulins containing 10 mg/ml.
After centrifugation, the radioactivity of the pellet corresponding
to Ang III bound to the antibody is measured. The technique used is
that described by GREENWOOD, F. C. et al. (Biochem. J., 1963, 89,
114-118).
Rabbit serum binds at most 60% of the iodine-125-labelled Ang
III.
F) Immunological characterisation of the anti-Ang III
antiserum.
a) Evaluation of the immunoreactivity of the antiserum prepared
with respect to Ang III.
100 .mu.l of a dilute solution of the anti-Ang III antiserum
(corresponding to approximately 50% of binding to iodinated Ang
III) are incubated in the presence of 50 .mu.l of free Ang III and
50 .mu.l of iodinated Ang III providing 5000 cpm for 18 hours at
4.degree. C. Separation of bound Ang III and free Ang III is
carried out by polyethylene glycol precipitation. The iodinated Ang
III-antibody binding is 50% displaced by adding 250 pg of Ang
III. The antibody hence recognises Ang III.
b) Cross-reactions of the anti-Ang III antibody with Ang I and Ang
II.
The cross-reaction of the anti-Ang III antibodies was measured with
respect to Ang I and Ang II. The percentage is less than 1% for
both peptides.
The anti-Ang III antibody may be used as obtained in the antiserum,
or after purification on protein A-Sepharose.
EXAMPLE 2
Capacity for capture of Ang III by the anti-Ang III antibodies
described above in the presence of anti-Ang II monoclonal antibody
4D8 and iodinated Ang II.
The antibody 4D8 is a mouse immunoglobulin; it is described in the
proceedings of the congress "The Renin-Angiotensin System as a
Therapeutic Target", Basle, 29-31 Oct. 1989, a SANOFI
communication.
A solution containing the following is prepared:
100 .mu.l of a solution of rabbit anti-Ang III antibody, obtained
in Example 1,
50 .mu.l of a solution of Ang III,
50 .mu.l of a solution of iodinated Ang II (specific activity 74
TBq/mmol (Amersham)), providing 4000 cpm,
100 .mu.l of a solution of anti-Ang II antibody (4D8), diluted in
such a way that it binds only 30% of the iodinated Ang II.
The reaction volume is made up to 400 .mu.l with the incubation
buffer, namely imidazole-HCl (0.1M; pH 7.4) containing 0.2% of BSA.
Incubation is carried out for 18 hours at +4.degree. C.
A suspension of 500 .mu.l of magnetic particles coated with
antibodies directed towards mouse immunoglobulins (Amerlex M,
marketed by Amersham) is added. Incubation is carried out for 15
minutes, the magnetic particles are then separated from the
reaction medium using a magnet and the radioactivity contained in
the magnetic phase is measured.
A series of measurements was carried out, varying the quantity of
Ang III from 0 to 40 pg/test. Control tests were performed in the
absence of anti-Ang III antibody.
Measurement of the radioactivity enables the results to be
expressed as the ratio of the concentrations B/Bo of the (iodinated
Ang II)-anti-Ang II antibody complex for each quantity of Ang
III.
These results are recorded in Table 1.
TABLE 1 ______________________________________ B/Bo (in p. cent) in
absence of in presence of Ang III anti-Ang III anti-Ang III
(pg/test) antibody antibody ______________________________________
0 100 100 5 67 100 10 39 100 20 18 100 40 5 96
______________________________________
As shown by the results in Table 1, the binding of iodinated Ang II
to the antibody 4D8 is decreased by adding Ang III; this binding is
fully restored by the presence of anti-Ang III antibody.
In this experiment, it was, in addition, verified that the addition
to the reaction medium of anti-Ang III antibody at the
concentration used has no influence on the reaction of Ang II with
the anti-Ang II antibodies.
EXAMPLE 3
Specific assay of Ang II in the presence of anti-Ang III
antibody.
According to one of the embodiments described above, the kit used
in this example comprises anti-Ang II monoclonal antibodies bound
to a solid support.
More precisely, the antibodies are bound to the walls of a tube
(Startube.RTM. marketed by NUNC). The kit then consists of the
following components:
NUNC Startube coated with anti-Ang II monoclonal antibodies 4D8 in
such a way that 30% of the iodinated Ang II is bound,
solutions of Ang II, for the calibration, calibrated with respect
to the Ang II international standard, batch 86/538, supplied by the
Medical Research Council,
a solution of Ang II labelled with iodine-125, of activity as
defined in Example 2,
a solution of rabbit anti-Ang III polyclonal antibody, prepared in
Example 1 above and diluted in such a way that it enables at least
10 pg of Ang III per test to be captured.
The assay is carried out in imidazole-HCl buffer (0.1M; pH 7.4)
containing 0.2% of BSA. The final reaction volume is 250 .mu.l.
a) Calibration curve in the presence of 10 pg of Ang III.
100 .mu.l of rabbit anti-Ang III polyclonal antibody are incubated
with 50 .mu.l of a solution of Ang III (corresponding to 10 pg of
Ang III), 50 .mu.l of a solution of Ang II standard and 50 .mu.l of
a solution of iodinated Ang II (4000 cpm) in the tube coated with
monoclonal antibody 4D8 for 18 hours at +4.degree. C.
The radioactivity is measured in the dry tubes after aspiration of
the incubation medium and washing with 1 ml of imidazole-HCl buffer
(0.1M; pH 7.4). The radioactivity is that of the anti-Ang II
antibody-iodinated Ang II complex.
A calibration series was prepared by varying the quantities of Ang
II from 0 to 40 pg/test. The measurement of radioactivity enables
the results to be expressed as the concentration ratio B/Bo of the
(iodinated Ang II)-anti-Ang II antibody complex for each quantity
of Ang II (column 1).
10 pg of Ang III are added in each of the tests corresponding to
the calibration curve (column 2), and the lowering of the ratio
B/Bo due to the cross-reaction of the anti-Ang II antibody with the
Ang III present is observed.
Lastly, in a third series of experiments (column 3, 10 pg of Ang
III and anti-Ang III antibody are added in each of the tests
corresponding to the calibration curve.
The results are recorded in Table 2 below.
TABLE 2 ______________________________________ Overload 10 pg Ang
III B/Bo (p. cent) Column 2 Column 3 Column 1 in absence of in
presence of Ang II B/Bo anti-Ang III anti-Ang III (pg/test) (p.
cent) antibody antibody ______________________________________ 0
100 23 100 1.25 84 21 86 2.5 81 22 83 5 67 22 66 10 47 18 49 20 29
12 36 40 17 11 21 ______________________________________
It is observed that the presence of anti-Ang III antibody enables
the values of the calibration curve to be recovered as a result of
the specific capture of the Ang III in the reaction medium.
It was, moreover, verified, as in Example 2, that the addition of
anti-Ang III antibody at the concentration used has no influence on
the Ang II calibration curve.
b) Specific assay of Ang II from plasma.
The actual assay is performed on a human plasma extract, the
extract being obtained according to the technique described by J.
NUSSBERGER et al. in Hypertension, 1985 (reference cited), and then
concentrated.
100 .mu.l of rabbit anti-Ang III polyclonal antibody are incubated
with 100 .mu.l of solution of Ang II standard or of plasma extract
to be assayed and 50 .mu.l of a solution of iodinated Ang II (4000
cpm) in the tube coated with monoclonal antibody 4D8 for 18 hours
at +4.degree. C.
The radioactivity is measured in the dry tubes after aspiration of
the incubation medium, as in Example 3 a).
A determination of the Ang II performed in the absence of anti-Ang
III antibody leads to the determination of an apparent quantity of
81 pg in the test, while, in the presence of anti-Ang III antibody,
only 57 pg are measured in the test. The difference results from
the removal of the quantity of Ang III immunocaptured by the
anti-Ang III antibodies.
It is deduced from this that the presence of Ang III leads in this
case to an error of ##EQU1## equivalent to 42%, in the direct
determination of Ang II carried out using anti-Ang II antibody
(4D8), as a result of the cross-reaction between this antibody and
the Ang III. This error may be eliminated by means of the
simultaneous use of the anti-Ang III antibody, which is one of the
subjects of the present invention.
EXAMPLE 4
Preparation of anti-Ang I antibody.
The peptide (VII) in which R.sub.2 =Ahx and R.sub.3 =Asp is
prepared, and the rabbit antiserum is then prepared by immunisation
according to the method described in Example 1.
EXAMPLE 5
Preparation of anti-hexapeptide antibody.
The peptide (VIII) in which R.sub.1 =Pro and R.sub.2 =Ahx is
prepared using the method described in Example 1, step A.
The antibody (rabbit antiserum) is then prepared using the method
described in Example 1, steps B, C and D, and thereafter
characterised.
EXAMPLE 6
Preparation of anti-pentapeptide antibody.
The procedure is as in the previous example for preparing the
peptide (IX) in which R.sub.1 =Pro and R.sub.2 =Ahx, and the rabbit
antiserum is then prepared by immunisation using the method
described in Example 1, and thereafter characterised.
EXAMPLE 7
Capacity for capture of pentapeptide by the anti-pentapeptide
antibodies prepared in Example 4, in the presence of anti-Ang II
4D8 and iodinated Ang II.
A solution containing the following is prepared:
100 .mu.l of a solution of anti-pentapeptide antibody, prepared in
Example 5,
100 .mu.l of a solution of pentapeptide,
50 .mu.l of a solution of iodinated Ang II (specific activity 74
TBq/mmol (Amersham)), providing 4000 cpm,
and is placed in a tube coated with antibody according to Example
3.
The final dilution of anti-pentapeptide antibody is 1/25.
Incubation is carried out for 18 hours at 4.degree. C. The
radioactivity is measured in the dry tubes after aspiration of the
incubation medium and washing with 1 ml of imidazole-HCl buffer
(0.1M; pH 7.4). The radioactivity is that of the anti-Ang II
antibody-iodinated Ang II complex.
The procedure is then as in Example 2, and the results recorded in
Table 3 below are obtained:
TABLE 3 ______________________________________ B/Bo (in p. cent) in
absence of in presence of Pentapeptide anti-pentapeptide
anti-peptapeptide (pg/test) antibody antibody
______________________________________ 0 100 100 1.25 95 100 2.5 92
100 5 75 100 10 69 100 20 48 97
______________________________________
EXAMPLE 8
Specific assay of Ang II in the presence of anti-Ang II antibody
and anti-pentapeptide antibody.
According to one of the embodiments described above, the kit used
in this example comprises anti-Ang II monoclonal antibodies bound
to a solid support.
More precisely, the antibodies are bound to the walls of a tube
(Startubex marketed by NUNC). The kit then consists of the
following components:
NUNC Startube coated with anti-Ang II monoclonal antibodies 4D8 in
such a way that 30% of the iodinated Ang II is bound,
solutions of Ang II, for the calibration, calibrated with respect
to the Ang II international standard, batch 86/538, supplied by the
Medical Research Council,
a solution of Ang II labelled with iodine-125, of activity as
defined in Example 2,
a solution of rabbit anti-Ang III polyclonal antibody, prepared in
Example 1 above and diluted in per test to be captured,
a solution of rabbit anti-peptapeptide polyclonal antibody prepared
in Example 5 above, such as to enable at least 10 pg of
pentapeptide per test to be captured.
The assay is carried out in imidazole-HCl buffer (0.1M; pH 7.4)
containing 0.2% of BSA. The final reaction volume is 250 .mu.l.
The calibration curve was established in the presence of 4 pg of
Ang III and 4 pg of pentapeptide.
100 .mu.l of a solution of rabbit anti-Ang III polyclonal antibody
and anti-pentapeptide are incubated with 50 .mu.l of a solution of
Ang III and pentapeptide (corresponding to 4 pg of each peptide),
50 .mu.l of a solution of Ang II standard and 50 .mu.l of a
solution of iodinated Ang II (4000 cpm) in the tube coated with
monoclonal antibody 4D8 for 18 hours at +4.degree. C.
The radioactivity is measured in the dry tubes after aspiration of
the incubation medium and washing with 1 ml of imidazole-HCl buffer
(0.1M; pH 7.4). The radioactivity is that of the anti-Ang II
antibody-iodinated Ang II complex.
A calibration series was prepared by varying the quantities of Ang
II from 0 to 40 pg/test. The measurement of radioactivity enables
the results to be expressed as the concentration ratio B/Bo of the
(iodinated Ang II)-anti-Ang II antibody complex for each quantity
of Ang II (column 1).
4 pg of Ang III and 4 pg of pentapeptide are added in each of the
tests corresponding to the calibration curve (column 2), and the
lowering of the ratio B/Bo due to the cross-reaction of the
anti-Ang II antibody with the Ang III and the pentapeptide present
is observed.
Lastly, in a third series of experiments (column 3), 4 pg of Ang
III, 4 pg of pentapeptide and the anti-Ang III and
anti-pentapeptide antibodies are added in each of the tests
corresponding to the calibration curve.
The results are recorded in Table 4 below:
TABLE 4 ______________________________________ Overload 4 pg Ang
III 4 pg Ang 4-8 B/Bo (p. cent) Column 2 Column 3 in absence of in
presence of Column 1 anti-Ang III and Anti-Ang III and Ang II B/Bo
anti-Ang 4-8 anti-Ang 4-8 (pg/test) (p. cent) antibodies antibodies
______________________________________ 0 100 53 100 2.5 85 47 87 5
72 44 73 10 56 34 55 20 38 27 38 40 22 19 25
______________________________________
It is observed that the presence of anti-Ang III and
anti-pentapeptide antibodies enables the values of the calibration
curve to be recovered as a result of the specific capture of the
Ang III and the pentapeptide in the reaction medium.
It was, moreover, verified, as in Example 2, that the addition of
anti-Ang III and anti-pentapeptide antibodies at the concentration
used has no influence on the Ang II calibration curve.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 3 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B)
CLONE: Angiotensin I (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AspArgValTyrIleHisProPheHisLeu 1510 (2) INFORMATION FOR SEQ ID
NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B)
TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(vii) IMMEDIATE SOURCE: (B) CLONE: Anti-Ang III antibody immunogen
(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 6..7 (D)
OTHER INFORMATION: /label=Xaa /note="A direct bond or chain-link
comprising 1 or 2 natural amino acids." (ix) FEATURE: (A) NAME/KEY:
Modified-site (B) LOCATION: 7..8 (D) OTHER INFORMATION: /label=Xaa
/note="A group of formula NH- (CH(sub)2)(sub)n-CO-, in which n
varies between 1 and 9." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ArgValTyrIleHisXaaXaaCys 15 (2) INFORMATION FOR SEQ ID NO:3: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (vii) IMMEDIATE SOURCE: (B) CLONE: Anti-Ang I
antibody immunogen (ix) FEATURE: (A) NAME/KEY: Modified-site (B)
LOCATION: 2..3 (D) OTHER INFORMATION: /label=Xaa /note="A group of
formula NH- (CH(sub)2)(sub)n-CO-, in w;hich n varies between 1 and
9." (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 3..4
(D) OTHER INFORMATION: /label=Xaa /note="A direct bond or a
chain-link comprising 1 or 2 natural amino acids." (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:3: CysX aaXaaArgValTyrIleHisProPheHisLeu
1510
* * * * *